Many experimental approaches have failed to find evidence for a scrapie- specific nucleic acid, yet the identification of distinct scrapie "strains" challenges this conclusion. Studies of prion strains have been facilitated by transgenic (Tg) mice since overexpression of PrP leads to a pronounced shortening of the incubation time. Use of mutated or chimeric PrP transgenes provides a source of PrP genetic variation not possible by other means. Syrian hamsters (SHa) have proven to offer significant advantages over mice for purification of infectious prions since SHa contain higher prion titers and 10-50-fold more PrPSc than mice making them more suitable for purification of PrPSc for structural studies. Tg mice expressing a chimeric SHa/Mo PrP gene designated MH2M are highly susceptible to prions derived from both mice and SHa. We propose to transfer distinct mouse prion strains into SHa using Tg(MH2MPrP) mice as an intermediate host. We have already made progress toward this goal and have obtained SHa(RML), SHa(Me7) and SHa(22A) prion inocula. We shall continue these studies by passaging of the 87V and 301V mouse strains from Tg(MH2MPrP) mice into SHa giving us five Mo- derived SHa prion isolates in addition to the SHa prion strains: Sc237, 139H, MT-C5, DY and Me7H. The strain Me7H is clearly distinct from Me7 derived from Tg(MH2MPrP) mice. The potential usefulness of these SHa strains will depend on several factors, including stability upon repeated passage, and our ability to assign discrete characteristics. Once two to more distinct SHa prion strains have been identified and large quantities of stock inocula have been prepared, we shall purify the prions and analyze the PrPSc using FTIR and CD spectroscopy, surface plasmon resonance, deuterium exchange as probed by mass spectrometry, chaotrope mediated denaturation in concert with bioassays, protease digestion and electron microscopy. Additional studies will also employ mass spectrometry in an effort to identify any additional components which might be responsible for scrapie prion diversity. To exploit further the advantages of the SHa, we plan to produce Tg SHa overexpressing SHaPrP and to derive SHa embryonic stem (ES) cells in which the endogenous SHaPrP gene has been disrupted. These ES cells will be used to produce chimeric SHa which will be crossed to produce PrP-o/o SHa. Such null SHa may be ideal hosts for expressing high levels of mutant and chimeric PrP transgenes, as well as genes derived from other species. To facilitate purification of PrPSc from Tg SHa and mice, affinity "tagged PrP" will be expressed once we show that it can be converted into PrPSc and supports prion replication. Deciphering the molecular basis of prion strains promises to open many avenues of research on prions as well as information transfer between proteins.